Enzyme immunoassays (EIA) and Western Blot assays (WB) have been routinely used for detecting HIV infection for several years. The implementation of these tests has significantly reduced the risk of transfusion-related HIV infection. However, some recent studies based on the detection of HIV DNA have demonstrated that some HIV infected individuals do not have any detectable amount of anti-HIV antibodies when the approved tests are performed. Data from several studies indicate that there is a "window period" estimated to span from a few weeks to several months or even several years between initial HIV infection and seroconversion. Cases of post-transfusion HIV infection have been reported from seronegative blood donors. This is assumed to be a consequence of donation during the "window" period on the part of these donors.
To verify the presence of HIV-antibody in donors of reactive samples to EIA, the Western Blot assay has been used for confirmatory testings (Ulstrup, J. C. et al, Lancet i:1151-1152). In this assay, 6 to 9 characteristic bands indicating antibodies to HIV surface and core antigens are observed if antibodies to HIV-1 proteins are present (positive), and no bands if antibodies are absent (negative). However, a significant proportion of EIA repeatedly reactive samples react only to the HIV-1 gag-derived core proteins (p17, p24 and p55) on the WB test (Kleinman, S., 1990, Arch. Pathol. Lab. Med. 114:298-303, Tribe D. E. et al, 1988, J. Clin. Microbiol. 26:641-647). This reactivity does not meet the definition of HIV-1 positive or negative in the criteria for WB tests and as a result these samples are labelled as HIV-1 WB indeterminates. Studies have established that 30 to 40 percent of EIA repeatedly positive donors are HIV-1 WB indeterminates, a figure that is typical in North America. Follow-up studies performed in regions of high prevalence of HIV show that while 95 to 99 percent of these are not infected with HIV, the remaining 1 to 5 percent of blood donors with HIV-1 WB indeterminate results are true seroconverters, usually at an early stage of infection (Busch, M. P. et al., 1991, Transfusion 31:129-137; Gallo D. et al, 1986, J. Clin. Microbiol. 23:1049-1051). In order to optimize the safety of transfusion, all donors with HIV-1 WB indeterminate results are deferred. The majority of donors with HIV-1 WB indeterminate results undergo needless anxiety, their deferral represents a significant loss of donors and recipients are still worried about contamination of blood taken from "window" period of seroconversion donors. A highly sensitive assay is thus also desirable in order to properly classify these indeterminate results.
Partially purified disrupted virus is used as an antigen for most currently licensed screening and confirmatory tests. Human cells are always used for culturing the HIV-1 virus (Dodd, R. Y. and C. T. Fang, 1990, Arch. Pathol. Lab. Med. 114:240-245). Recombinant proteins and synthetic peptides have been recently licensed for screening tests (Busch M. P. et al., 1991, Transfusion 31:129-137; Das P.C. et al., 1992, Trans Med 2:249-250; Ramirez E. P., 1992, J. Clin. Microbiol. 30:801-805). In theory, these antigens can provide more sensitive and definitive assays. However, most recombinant proteins are produced in E. coli and denatured during the purification and processing of the antigens. Also, a certain proportion of donors still show cross-reactivity to HIV core antigens (such as antigen p24), and sensitivity is limited to detecting very early HIV antibodies.
A serious drawback to the use of synthetic peptides is related to the fact that in some HIV-1 infected patients and seroconverters in high risk populations, the serum antibody titre is very low, or undetectable, either because of complex formation between p24 and antibodies or the loss of specific clones of antibody producing cells (Orsknov, L. B., Eur. J. Clin. Microbiol. Inf. Dis. 8:614). Synthetic peptides, which in general only cover one or two epitopes, do not efficiently detect such low titre antibodies and furthermore have a limited ability to take on the natural three dimensional structures of antigen.
An immunofluorescence assay (IFA) has recently been licensed as an alternate confirmatory test for detecting HIV-1 antibodies (FDA Memorandum, 1992, Summer:56-67). IFA is rapid, simple and inexpensive. However, it is a subjective procedure requiring well trained personnel (Ascher, M. S. 1990, Arch. Pathol. Lab. Med. 114:246-248). This is a limitation for users and false positive and false negative results still occur in the IFA tests because of cross-reactivity of some antibodies to antigens on the human cells in which the HIV virus is cultured. Fixing of the infected cells before incubation also has been shown to lead to false positive and negative results (McHugh, T. M., 1986, Diagnos. Immunol. 4:233-240).
An assay based on immunofluorescence was recently developed to detect antibodies to HIV-1 by using flow cytometry (FIFA) (Sligh, J. M., 1989, Am. J. Clin. Path. 91:210-214). FIFA is a sensitive, quantitative test. In a typical FIFA protocol, HIV-1 infected cells are used directly for the test. However, false positive and false negative results may occur because of the HIV-1 antibody cross-reactivity to the antigens (such as HLA) on human cells in which the virus is cultured and used as antigens in FIFA. Another concern is with biohazards caused by the infectious virus which is a big limitation for users. Fixing the HIV-1 infected cells for inactivation of the virus before incubation has shown to lead to higher cross reactivity on the cells.
The present invention is directed to a flow cytometric immunofluorescence assay (r-FIFA) using insoluble forms of recombinant proteins expressed in an expression system such as the baculovirus system. In a preferred embodiment, r-FIFA is used for the detection of the HIV virus infection. Insoluble forms of recombinant HIV-1 proteins such as HIV-1 gag p45 protein, gag gp-41 chimeric proteins, HIV-1 precursor polyproteins pol 97 and gp160 are used as autologous carriers (in place of beads) and antigens to detect HIV-1 antibodies using flow cytometry. The baculovirus expression system has become a major recombinant protein production system because of several advantages over bacterial and mammalian systems including superior yield of recombinant protein, safety (baculovirus is not infectious to humans) and fidelity of its products.
In sensitivity comparison between r-FIFA and currently licensed tests, r-FIFA was found to be more sensitive, the average increase in sensivity for early detection of HIV-1 infection being greater than 20 days. r-FIFA has permitted the detection and quantification of HIV-1 specific IgG, IgM and IgA antibodies during the window period. The use of HIV-1 recombinant proteins in an immunofluorescence assay (r-FIFA) solves the problems of antibody cross-reactivity to antigen on human cells and the biohazard concern with the original FIFA.